RESUMEN
The arsenic-specific ACR3 transporter plays pivotal roles in As detoxification in yeast and a group of ancient tracheophytes, the ferns. Despite putative ACR3 genes being present in the genomes of bryophytes, whether they have the same relevance also in this lineage is currently unknown. In this study, we characterized the MpACR3 gene from the bryophyte Marchantia polymorpha L. through a multiplicity of functional approaches ranging from phylogenetic reconstruction, expression analysis, loss- and gain-of-function as well as genetic complementation with an MpACR3 gene tagged with a fluorescent protein. Genetic complementation demonstrates that MpACR3 plays a pivotal role in As tolerance in M. polymorpha, with loss-of-function Mpacr3 mutants being hypersensitive and MpACR3 overexpressors more tolerant to As. Additionally, MpACR3 activity regulates intracellular As concentration, affects its speciation and controls the levels of intracellular oxidative stress. The MpACR3::3xCitrine appears to localize at the plasma membrane and possibly in other endomembrane systems. Taken together, these results demonstrate the pivotal function of ACR3 detoxification in both sister lineages of land plants, indicating that it was present in the common ancestor to all embryophytes. We propose that Mpacr3 mutants could be used in developing countries as low-cost and low-technology visual bioindicators to detect As pollution in water.
Asunto(s)
Arsénico , Marchantia , Marchantia/genética , Marchantia/metabolismo , Marchantia/efectos de los fármacos , Arsénico/toxicidad , Arsénico/metabolismo , Inactivación Metabólica , Filogenia , Estrés Oxidativo/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Even though stable genomic transformation of sporelings and thalli of Marchantia polymorpha is straightforward and efficient, numerous problems can arise during critical phases of the process such as efficient spore production, poor selection capacity of antibiotics or low transformation efficiency. It is therefore also desirable to establish quick methods not relying on stable transgenics to analyze the localization, interactions and functions of proteins of interest. The introduction of foreign DNA into living cells via biolistic mechanisms has been first reported roughly 30 years ago and has been commonly exploited in established plant model species such as Arabidopsis thaliana or Nicotiana benthamiana. Here, we report the fast and reliable transient biolistic transformation of Marchantia thallus epidermal cells using fluorescent protein fusions. We present a catalog of fluorescent markers which can be readily used for tagging of a variety of subcellular compartments. Moreover, we report the functionality of the bimolecular fluorescence complementation (BiFC) in M. polymorpha with the example of the p-body markers MpDCP1/2. Finally, we provide standard staining procedures for live cell imaging in M. polymorpha, applicable to visualize cell boundaries or cellular structures, to complement or support protein localizations and to understand how results gained by transient transformations can be embedded in cell architecture and dynamics. Taken together, we offer a set of easy and quick tools for experiments that aim at understanding subcellular localization, protein-protein interactions and thus functions of proteins of interest in the emerging early diverging land plant model M. polymorpha.
RESUMEN
Plant cell growth requires the coordinated expansion of the protoplast and the cell wall, which is controlled by an elaborate system of cell wall integrity (CWI) sensors linking the different cellular compartments. LRR-eXtensins (LRXs) are cell wall-attached extracellular regulators of cell wall formation and high-affinity binding sites for RALF (Rapid ALkalinization Factor) peptide hormones that trigger diverse physiological processes related to cell growth. LRXs function in CWI sensing and in the case of LRX4 of Arabidopsis thaliana, this activity was shown to involve interaction with the transmembrane Catharanthus roseus Receptor-Like Kinase1-Like (CrRLK1L) protein FERONIA (FER). Here, we demonstrate that binding of RALF1 and FER is common to most tested LRXs of vegetative tissue, including LRX1, the main LRX protein of root hairs. Consequently, an lrx1-lrx5 quintuple mutant line develops shoot and root phenotypes reminiscent of the fer-4 knock-out mutant. The previously observed membrane-association of LRXs, however, is FER-independent, suggesting that LRXs bind not only FER but also other membrane-localized proteins to establish a physical link between intra- and extracellular compartments. Despite evolutionary diversification of various LRX proteins, overexpression of several chimeric LRX constructs causes cross-complementation of lrx mutants, indicative of comparable functions among members of this protein family. Suppressors of the pollen-growth defects induced by mutations in the CrRLK1Ls ANXUR1/2 also alleviate lrx1 lrx2-induced mutant root hair phenotypes. This suggests functional similarity of LRX-CrRLK1L signaling processes in very different cell types and indicates that LRX proteins are components of conserved processes regulating cell growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Pared Celular/metabolismo , Hormonas Peptídicas/metabolismo , Fosfotransferasas/metabolismo , Proteínas de Arabidopsis/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Genes de Plantas , Mutación , Fosfotransferasas/genética , Raíces de Plantas/citología , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Polen/citología , Polen/crecimiento & desarrollo , Dominios Proteicos/genética , Mapas de Interacción de Proteínas , Plantones/citología , Plantones/crecimiento & desarrollo , Transducción de Señal/genéticaRESUMEN
Rooting cells and pollen tubes-key adaptative innovations that evolved during the colonization and subsequent radiation of plants on land-expand by tip growth. Tip growth relies on a tight coordination between the protoplast growth and the synthesis/remodeling of the external cell wall. In root hairs and pollen tubes of the seed plant Arabidopsis thaliana, cell wall integrity (CWI) mechanisms monitor this coordination through the Malectin-like receptor kinases (MLRs), such as AtANXUR1 and AtFERONIA, that act upstream of the AtMARIS PTI1-like kinase. Here, we show that rhizoid growth in the early diverging plant, Marchantia polymorpha, is also controlled by an MLR and PTI1-like signaling module. Rhizoids, root hairs, and pollen tubes respond similarly to disruption of MLR and PTI1-like encoding genes. Thus, the MLR and PTI1-like signaling module that controls CWI during tip growth is conserved between M. polymorpha and A. thaliana, suggesting that it was active in the common ancestor of land plants.
Asunto(s)
Meristema/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Tubo Polínico/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Evolución Biológica , Pared Celular/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Genes de Plantas/genética , Hepatophyta/genética , Hepatophyta/metabolismo , Meristema/genética , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Tubo Polínico/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , Transducción de Señal , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Fast tip-growing plant cells such as pollen tubes (PTs) and root hairs (RHs) require a robust coordination between their internal growth machinery and modifications of their extracellular rigid, yet extensible, cell wall (CW). Part of this essential coordination is governed by members of the Catharanthus roseus receptor-like kinase1-like (CrRLK1L) subfamily of RLKs with FERONIA (FER) and its closest homologs, ANXUR1 (ANX1) and ANX2, controlling CW integrity during RH and PT growth, respectively. Recently, Leucine-Rich Repeat Extensin 8 (LRX8) to LRX11 were also shown to be important for CW integrity in PTs. We previously reported an anx1 anx2 suppressor screen in Arabidopsis thaliana that revealed MARIS (MRI) as a positive regulator of both FER- and ANX1/2-dependent CW integrity pathways. Here, we characterize a suppressor that exhibits a weak rescue of the anx1 anx2 PT bursting phenotype and a short RH phenotype. The corresponding suppressor mutation causes a D94N substitution in a Type One Protein Phosphatase we named ATUNIS1 (AUN1). We show that AUN1 and its closest homolog, AUN2, are nucleocytoplasmic negative regulators of tip growth. Moreover, we demonstrate that AUN1D94N and AUN1H127A harboring mutations in key amino acids of the conserved catalytic site of phosphoprotein phosphatases function as dominant amorphic variants that repress PT growth. Finally, genetic interaction studies using the hypermorph MRIR240C and amorph AUN1D94N dominant variants indicate that LRX8-11 and ANX1/2 function in distinct but converging pathways to fine-tune CW integrity during tip growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Pared Celular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Raíces de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Pared Celular/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Mutación/genética , Fosfoproteínas Fosfatasas/genética , Raíces de Plantas/genéticaRESUMEN
Plant cells are surrounded by cell walls protecting them from a myriad of environmental challenges. For successful habitat adaptation, extracellular cues are perceived at the cell wall and relayed to downstream signaling constituents to mediate dynamic cell wall remodeling and adapted intracellular responses. Plant malectin-like receptor kinases, also known as Catharanthus roseus receptor-like kinase 1-like proteins (CrRLK1Ls), take part in these perception and relay processes. CrRLK1Ls are involved in many different plant functions. Their ligands, interactors, and downstream signaling partners are being unraveled, and studies about CrRLK1Ls' roles in plant species other than the plant model Arabidopsis thaliana are beginning to flourish. This review focuses on recent CrRLK1L-related advances in cell growth, reproduction, hormone signaling, abiotic stress responses, and, particularly, immunity. We also give an overview of the comparative genomics and evolution of CrRLK1Ls, and present a brief outlook for future research.
Asunto(s)
Pared Celular/metabolismo , Inmunidad de la Planta , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Estrés FisiológicoRESUMEN
The communication of changes in the extracellular matrix to the interior of the cell is crucial for a cell's function. The extracellular peptides of the RAPID ALKALINIZATION FACTOR (RALF) family have been identified as ligands of receptor-like kinases of the CrRLK1L subclass, but the exact mechanism of their perception is unclear. We found that Arabidopsis RALF4 and RALF19 redundantly regulate pollen tube integrity and growth, and that their function depends on pollen-expressed proteins of the LEUCINE-RICH REPEAT EXTENSIN (LRX) family, which play a role in cell wall development but whose mode of action is not understood. The LRX proteins interact with RALFs, monitoring cell wall changes, which are communicated to the interior of the pollen tube via the CrRLK1L pathway to sustain normal growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas Portadoras/metabolismo , Tubo Polínico/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Pared Celular/metabolismo , Péptidos/genética , Péptidos/metabolismo , Tubo Polínico/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
Plants have evolved two tiers of immune receptors to detect infections: cell surface-resident pattern recognition receptors (PRRs) that sense microbial signatures and intracellular nucleotide binding domain leucine-rich repeat (NLR) proteins that recognize pathogen effectors. How PRRs and NLRs interconnect and activate the specific and overlapping plant immune responses remains elusive. A genetic screen for components controlling plant immunity identified ANXUR1 (ANX1), a malectin-like domain-containing receptor-like kinase, together with its homolog ANX2, as important negative regulators of both PRR- and NLR-mediated immunity in Arabidopsis thaliana ANX1 constitutively associates with the bacterial flagellin receptor FLAGELLIN-SENSING2 (FLS2) and its coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). Perception of flagellin by FLS2 promotes ANX1 association with BAK1, thereby interfering with FLS2-BAK1 complex formation to attenuate PRR signaling. In addition, ANX1 complexes with the NLR proteins RESISTANT TO PSEUDOMONAS SYRINGAE2 (RPS2) and RESISTANCE TO P. SYRINGAE PV MACULICOLA1. ANX1 promotes RPS2 degradation and attenuates RPS2-mediated cell death. Surprisingly, a mutation that affects ANX1 function in plant immunity does not disrupt its function in controlling pollen tube growth during fertilization. Our study thus reveals a molecular link between PRR and NLR protein complexes that both associate with cell surface-resident ANX1 and uncovers uncoupled functions of ANX1 and ANX2 during plant immunity and sexual reproduction.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Regulación de la Expresión Génica de las Plantas , Inmunidad de la Planta/genética , Proteínas Quinasas/metabolismo , Alarminas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Resistencia a la Enfermedad/efectos de los fármacos , Flagelina/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes Reporteros , Luciferasas/metabolismo , Mutación/genética , Inmunidad de la Planta/efectos de los fármacos , Plantas Modificadas Genéticamente , Tubo Polínico/efectos de los fármacos , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Quinasas/genética , Pseudomonas syringae/efectos de los fármacos , Pseudomonas syringae/patogenicidad , Receptores de Reconocimiento de Patrones/metabolismo , Reproducción/efectos de los fármacos , Virulencia/efectos de los fármacosRESUMEN
While cytosolic calcium (Ca2+) plays a central role in a myriad of signaling pathways as a secondary messenger, how dynamic changes of cytosolic calcium relate to cell growth control remains poorly understood. The engineering and continuous improvements of genetically encoded calcium sensors such as the Yellow Cameleon (YC) sensors combined with advances in microscopy have allowed imaging with great resolution of the spatiotemporal characteristics of cytosolic [Ca2+]cyt in individual cells. An exciting new step consists therefore in cautiously studying calcium dynamics in mutant backgrounds that display disturbed cellular growth behavior to further enhance our understanding on growth-related processes. Here, we describe methods to perform imaging of [Ca2+]cyt dynamics in growing Arabidopsis thaliana wild-type and NADPH-oxidase deficient rbohH rbohJ pollen tubes stably expressing YC3.6 using confocal laser scanning microscopy. We also present different ways to extract meaningful qualitative and quantitative information about calcium dynamics during growth.
Asunto(s)
Citosol/metabolismo , Microscopía Confocal/métodos , NADPH Oxidasas/metabolismo , Tubo Polínico/metabolismo , Arabidopsis/metabolismo , Calcio/metabolismo , Pared Celular/metabolismoRESUMEN
Growing plant cells need to rigorously coordinate external signals with internal processes. For instance, the maintenance of cell wall (CW) integrity requires the coordination of CW sensing with CW remodeling and biosynthesis to avoid growth arrest or integrity loss. Despite the involvement of receptor-like kinases (RLKs) of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily and the reactive oxygen species-producing NADPH oxidases, it remains largely unknown how this coordination is achieved. ANXUR1 (ANX1) and ANX2, two redundant members of the CrRLK1L subfamily, are required for tip growth of the pollen tube (PT), and their closest homolog, FERONIA, controls root-hair tip growth. Previously, we showed that ANX1 overexpression mildly inhibits PT growth by oversecretion of CW material, whereas pollen tubes of anx1 anx2 double mutants burst spontaneously after germination. Here, we report the identification of suppressor mutants with improved fertility caused by the rescue of anx1 anx2 pollen tube bursting. Mapping of one these mutants revealed an R240C nonsynonymous substitution in the activation loop of a receptor-like cytoplasmic kinase (RLCK), which we named MARIS (MRI). We show that MRI is a plasma membrane-localized member of the RLCK-VIII subfamily and is preferentially expressed in both PTs and root hairs. Interestingly, mri-knockout mutants display spontaneous PT and root-hair bursting. Moreover, expression of the MRI(R240C) mutant, but not its wild-type form, partially rescues the bursting phenotypes of anx1 anx2 PTs and fer root hairs but strongly inhibits wild-type tip growth. Thus, our findings identify a novel positive component of the CrRLK1L-dependent signaling cascade that coordinates CW integrity and tip growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Catharanthus/enzimología , Citoplasma/enzimología , Raíces de Plantas/enzimología , Tubo Polínico/enzimología , Proteínas Quinasas/metabolismo , Proteínas Tirosina Fosfatasas Similares a Receptores/metabolismo , Transducción de Señal/fisiología , Procesamiento de Imagen Asistido por Computador , Microscopía de Interferencia , Raíces de Plantas/crecimiento & desarrollo , Tubo Polínico/crecimiento & desarrolloRESUMEN
Pollen tube (PT) reception in flowering plants describes the crosstalk between the male and female gametophytes upon PT arrival at the synergid cells of the ovule. It leads to PT growth arrest, rupture, and sperm cell release, and is thus essential to ensure double fertilization. Here, we describe TURAN (TUN) and EVAN (EVN), two novel members of the PT reception pathway that is mediated by the FERONIA (FER) receptor-like kinase (RLK). Like fer, mutations in these two genes lead to PT overgrowth inside the female gametophyte (FG) without PT rupture. Mapping by next-generation sequencing, cytological analysis of reporter genes, and biochemical assays of glycoproteins in RNAi knockdown mutants revealed both genes to be involved in protein N-glycosylation in the endoplasmic reticulum (ER). TUN encodes a uridine diphosphate (UDP)-glycosyltransferase superfamily protein and EVN a dolichol kinase. In addition to their common role during PT reception in the synergids, both genes have distinct functions in the pollen: whereas EVN is essential for pollen development, TUN is required for PT growth and integrity by affecting the stability of the pollen-specific FER homologs ANXUR1 (ANX1) and ANX2. ANX1- and ANX2-YFP reporters are not expressed in tun pollen grains, but ANX1-YFP is degraded via the ER-associated degradation (ERAD) pathway, likely underlying the anx1/2-like premature PT rupture phenotype of tun mutants. Thus, as in animal sperm-egg interactions, protein glycosylation is essential for the interaction between the female and male gametophytes during PT reception to ensure fertilization and successful reproduction.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Glicosiltransferasas/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Tubo Polínico , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Glicosilación , MutaciónRESUMEN
It has become increasingly apparent that the extracellular matrix (ECM), which in plants corresponds to the cell wall, can influence intracellular activities in ways that go far beyond their supposedly passive mechanical support. In plants, growing cells use mechanisms sensing cell wall integrity to coordinate cell wall performance with the internal growth machinery to avoid growth cessation or loss of integrity. How this coordination precisely works is unknown. Previously, we reported that in the tip-growing pollen tube the ANXUR receptor-like kinases (RLKs) of the CrRLK1L subfamily are essential to sustain growth without loss of cell wall integrity in Arabidopsis. Here, we show that over-expression of the ANXUR RLKs inhibits growth by over-activating exocytosis and the over-accumulation of secreted cell wall material. Moreover, the characterization of mutations in two partially redundant pollen-expressed NADPH oxidases coupled with genetic interaction studies demonstrate that the ANXUR RLKs function upstream of these NADPH oxidases. Using the H2O2-sensitive HyPer and the Ca²âº-sensitive YC3.60 sensors in NADPH oxidase-deficient mutants, we reveal that NADPH oxidases generate tip-localized, pulsating H2O2 production that functions, possibly through Ca²âº channel activation, to maintain a steady tip-focused Ca²âº gradient during growth. Our findings support a model where ECM-sensing receptors regulate reactive oxygen species production, Ca²âº homeostasis, and exocytosis to coordinate ECM-performance with the internal growth machinery.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/enzimología , NADPH Oxidasas/genética , Tubo Polínico/enzimología , Proteínas Quinasas/fisiología , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Calcio/metabolismo , Pared Celular/enzimología , Exocitosis , Matriz Extracelular/metabolismo , Germinación , Homeostasis , Peróxido de Hidrógeno/metabolismo , NADPH Oxidasas/metabolismo , Infertilidad Vegetal , Tubo Polínico/citología , Tubo Polínico/crecimiento & desarrolloRESUMEN
Plant cell expansion is controlled by a fine-tuned balance between intracellular turgor pressure, cell wall loosening and cell wall biosynthesis. To understand these processes, it is important to gain in-depth knowledge of cell wall mechanics. Pollen tubes are tip-growing cells that provide an ideal system to study mechanical properties at the single cell level. With the available approaches it was not easy to measure important mechanical parameters of pollen tubes, such as the elasticity of the cell wall. We used a cellular force microscope (CFM) to measure the apparent stiffness of lily pollen tubes. In combination with a mechanical model based on the finite element method (FEM), this allowed us to calculate turgor pressure and cell wall elasticity, which we found to be around 0.3 MPa and 20-90 MPa, respectively. Furthermore, and in contrast to previous reports, we showed that the difference in stiffness between the pollen tube tip and the shank can be explained solely by the geometry of the pollen tube. CFM, in combination with an FEM-based model, provides a powerful method to evaluate important mechanical parameters of single, growing cells. Our findings indicate that the cell wall of growing pollen tubes has mechanical properties similar to rubber. This suggests that a fully turgid pollen tube is a relatively stiff, yet flexible cell that can react very quickly to obstacles or attractants by adjusting the direction of growth on its way through the female transmitting tissue.
Asunto(s)
Lilium/fisiología , Células Vegetales/fisiología , Tubo Polínico/fisiología , Fenómenos Biomecánicos , Pared Celular/fisiología , Simulación por Computador , Elasticidad , Lilium/anatomía & histología , Microscopía/instrumentación , Microscopía/métodos , Modelos Biológicos , Tubo Polínico/anatomía & histología , Presión , Estrés MecánicoRESUMEN
In plants, receptor-like kinases regulate many processes during reproductive and vegetative development. The Arabidopsis subfamily of Catharanthus roseus RLK1-like kinases (CrRLK1Ls) comprises 17 members with a putative extracellular carbohydrate-binding malectin-like domain. Only little is known about the functions of these proteins, although mutant analyses revealed a role during cell elongation, polarized growth, and fertilization. However, the molecular nature of the underlying signal transduction cascades remains largely unknown. CrRLK1L proteins are also involved in biotic and abiotic stress responses. It is likely that carbohydrate-rich ligands transmit a signal, which could originate from cell wall components, an arriving pollen tube, or a pathogen attack. Thus, post-translational modifications could be crucial for CrRLK1L signal transduction and ligand binding.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Comunicación Celular , Pared Celular/enzimología , Fosfotransferasas/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Aumento de la Célula , Polaridad Celular , Pared Celular/inmunología , Fosfotransferasas/genética , Células Vegetales/enzimología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Tubo Polínico/citología , Tubo Polínico/enzimología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Pseudomonas syringae/inmunología , Pseudomonas syringae/patogenicidad , Estrés Fisiológico , Transcripción GenéticaRESUMEN
Successful pollination depends on cell-cell communication and rapid cellular responses. In Arabidopsis, the pollen grain lands on a dry stigma, where it hydrates, germinates and grows a pollen tube that delivers the sperm cells to the female gametophyte to effect double fertilization. Various studies have emphasized that a mature, dehydrated pollen grain contains all the transcripts and proteins required for germination and initial pollen tube growth. Therefore, it is important to explore the role of post-translational modifications (here phosphorylation), through which many processes induced by pollination are probably controlled. We report here a phosphoproteomic study conducted on mature Arabidopsis pollen grains with the aim of identifying potential targets of phosphorylation. Using three enrichment chromatographies, a broad coverage of pollen phosphoproteins with 962 phosphorylated peptides corresponding to 598 phosphoproteins was obtained. Additionally, 609 confirmed phosphorylation sites were successfully mapped. Two hundred and seven of 240 phosphoproteins that were absent from the PhosPhAt database containing the empirical Arabidopsis phosphoproteome showed highly enriched expression in pollen. Gene ontology (GO) enrichment analysis of these 240 phosphoproteins shows an over-representation of GO categories crucial for pollen tube growth, suggesting that phosphorylation regulates later processes of pollen development. Moreover, motif analyses of pollen phosphopeptides showed an over-representation of motifs specific for Ca²âº/calmodulin-dependent protein kinases, mitogen-activated protein kinases, and binding motifs for 14-3-3 proteins. Lastly, one tyrosine phosphorylation site was identified, validating the TDY dual phosphorylation motif of mitogen-activated protein kinases (MPK8/MPK15). This study provides a solid basis to further explore the role of phosphorylation during pollen development.
Asunto(s)
Arabidopsis/metabolismo , Fosfoproteínas/metabolismo , Polen/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma , Proteínas 14-3-3/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Bases de Datos Genéticas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos , Fosfopéptidos/química , Fosfopéptidos/aislamiento & purificación , Fosfopéptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/aislamiento & purificación , Fosforilación , Polen/química , Polen/crecimiento & desarrollo , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Polinización , TranscriptomaRESUMEN
In plants, organ formation and cell elongation require the constant adjustment of the dynamic and adaptable cell wall in response to environmental cues as well as internal regulators, such as light, mechanical stresses, pathogen attacks, phytohormones, and other signaling molecules. The molecular mechanisms that perceive these cues and translate them into cellular responses to maintain integrity and remodelling of the carbohydrate-rich cell wall for the coordination of cell growth are still poorly understood. In the last 3 years, the function of six membrane-localized receptor-like kinases (RLKs) belonging to the CrRLK1L family has been linked to the control of cell elongation in vegetative and reproductive development. Moreover, the presence of putative carbohydrate-binding domains in the extracellular domains of these CrRLK1Ls makes this receptor family an excellent candidate for coordinating cell growth, cell-cell communication, and constant cell wall remodelling during the plant life cycle.
Asunto(s)
Comunicación Celular/fisiología , Pared Celular/metabolismo , Desarrollo de la Planta , Proteínas Quinasas/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Catharanthus/enzimología , Catharanthus/genética , Catharanthus/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Fosfotransferasas/clasificación , Fosfotransferasas/genética , Fosfotransferasas/fisiología , Filogenia , Plantas/enzimología , Plantas/genética , Proteínas Quinasas/clasificación , Proteínas Quinasas/genética , Receptores de Superficie Celular/clasificación , Receptores de Superficie Celular/genéticaRESUMEN
The continuing rise in atmospheric CO2 causes stomatal pores in leaves to close and thus globally affects CO2 influx into plants, water use efficiency and leaf heat stress. However, the CO2-binding proteins that control this response remain unknown. Moreover, which cell type responds to CO2, mesophyll or guard cells, and whether photosynthesis mediates this response are matters of debate. We demonstrate that Arabidopsis thaliana double-mutant plants in the beta-carbonic anhydrases betaCA1 and betaCA4 show impaired CO2-regulation of stomatal movements and increased stomatal density, but retain functional abscisic-acid and blue-light responses. betaCA-mediated CO2-triggered stomatal movements are not, in first-order, linked to whole leaf photosynthesis and can function in guard cells. Furthermore, guard cell betaca-overexpressing plants exhibit instantaneous enhanced water use efficiency. Guard cell expression of mammalian alphaCAII complements the reduced sensitivity of ca1 ca4 plants, showing that carbonic anhydrase-mediated catalysis is an important mechanism for betaCA-mediated CO2-induced stomatal closure and patch clamp analyses indicate that CO2/HCO3- transfers the signal to anion channel regulation. These findings, together with ht1-2 (ref. 9) epistasis analysis demonstrate that carbonic anhydrases function early in the CO2 signalling pathway, which controls gas-exchange between plants and the atmosphere.
Asunto(s)
Arabidopsis/metabolismo , Dióxido de Carbono/farmacología , Anhidrasa Carbónica IV/metabolismo , Anhidrasa Carbónica I/metabolismo , Canales Iónicos/metabolismo , Estomas de Plantas/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Anhidrasa Carbónica I/genética , Anhidrasa Carbónica IV/genética , Prueba de Complementación Genética , Activación del Canal Iónico , Luz , Fotosíntesis , Reguladores del Crecimiento de las Plantas/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Estomas de Plantas/efectos de los fármacos , Estomas de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Agua/metabolismoRESUMEN
The precise delivery of male to female gametes during reproduction in eukaryotes requires complex signal exchanges and a flawless communication between male and female tissues. In angiosperms, molecular mechanisms have recently been revealed that are crucial for the dialog between male (pollen tube) and female gametophytes required for successful sperm delivery. When pollen tubes reach the female gametophyte, they arrest growth, burst and discharge their sperm cells. These processes are under the control of the female gametophyte via the receptor-like serine-threonine kinase (RLK) FERONIA (FER). However, the male signaling components that control the sperm delivery remain elusive. Here, we show that ANXUR1 and ANXUR2 (ANX1, ANX2), which encode the closest homologs of the FER-RLK in Arabidopsis, are preferentially expressed in pollen. Moreover, ANX1-YFP and ANX2-YFP fusion proteins display polar localization to the plasma membrane at the tip of the pollen tube. Finally, genetic analyses demonstrate that ANX1 and ANX2 function redundantly to control the timing of pollen tube discharge as anx1 anx2 double-mutant pollen tubes cease their growth and burst in vitro and fail to reach the female gametophytes in vivo. We propose that ANX-RLKs constitutively inhibit pollen tube rupture and sperm discharge at the tip of growing pollen tubes to sustain their growth within maternal tissues until they reach the female gametophytes. Upon arrival, the female FER-dependent signaling cascade is activated to mediate pollen tube reception and fertilization, while male ANX-dependent signaling is deactivated, enabling the pollen tube to rupture and deliver its sperm cells to effect fertilization.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Arabidopsis/fisiología , Fosfotransferasas/genética , Fosfotransferasas/fisiología , Arabidopsis/crecimiento & desarrollo , Secuencia de Bases , ADN de Plantas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Mutagénesis Insercional , Fenotipo , Plantas Modificadas Genéticamente , Polen/genética , Polen/crecimiento & desarrollo , Polen/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reproducción , Transducción de Señal , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismoRESUMEN
In eukaryotes, fertilization relies on complex and specialized mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union [1-4]. In flowering plants, the haploid male gametophyte or pollen tube (PT) [5] carries two nonmotile sperm cells to the female gametophyte (FG) or embryo sac [6] during a long assisted journey through the maternal tissues [7-10]. In Arabidopsis, typically one PT reaches one of the two synergids of the FG (Figure 1A), where it terminates its growth and delivers the sperm cells, a poorly understood process called pollen-tube reception. Here, we report the isolation and characterization of the Arabidopsis mutant abstinence by mutual consent (amc). Interestingly, pollen-tube reception is impaired only when an amc pollen tube reaches an amc female gametophyte, resulting in pollen-tube overgrowth and completely preventing sperm discharge and the development of homozygous mutants. Moreover, we show that AMC is strongly and transiently expressed in both male and female gametophytes during fertilization and that AMC functions in gametophytes as a peroxin essential for protein import into peroxisomes. These findings show that peroxisomes play an unexpected key role in gametophyte recognition and implicate a diffusible signal emanating from either gametophyte that is required for pollen-tube discharge.