RESUMO
Phosphatidic acid (PA) and reactive oxygen species (ROS) are crucial cellular messengers mediating diverse signaling processes in metazoans and plants. How PA homeostasis is tightly regulated and intertwined with ROS signaling upon immune elicitation remains elusive. We report here that Arabidopsis diacylglycerol kinase 5 (DGK5) regulates plant pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). The pattern recognition receptor (PRR)-associated kinase BIK1 phosphorylates DGK5 at Ser-506, leading to a rapid PA burst and activation of plant immunity, whereas PRR-activated intracellular MPK4 phosphorylates DGK5 at Thr-446, which subsequently suppresses DGK5 activity and PA production, resulting in attenuated plant immunity. PA binds and stabilizes the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), regulating ROS production in plant PTI and ETI, and their potentiation. Our data indicate that distinct phosphorylation of DGK5 by PRR-activated BIK1 and MPK4 balances the homeostasis of cellular PA burst that regulates ROS generation in coordinating two branches of plant immunity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Diacilglicerol Quinase , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Diacilglicerol Quinase/metabolismo , NADPH Oxidases/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosforilação , Imunidade Vegetal , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Phospholipase C (PLC) has been implicated in several stress responses, including drought. Overexpression (OE) of PLC has been shown to improve drought tolerance in various plant species. Arabidopsis contains nine PLC genes, subdivided into four clades. Earlier, OE of PLC3, -5 or -7 were found to increase Arabidopsis' drought tolerance. Here, we confirm this for three other PLCs: PLC2, the only constitutively expressed AtPLC; PLC4, reported to have reduced salt tolerance; and PLC9, of which the encoded enzyme was presumed to be catalytically inactive. To compare each PLC and to discover any other potential phenotype, two independent OE lines of six AtPLC genes, representing all four clades, were simultaneously monitored with the GROWSCREEN FLUORO phenotyping platform, under both control- and mild drought conditions. To investigate which tissues were most relevant to achieve drought survival, we additionally expressed AtPLC5 using 13 different cell- or tissue-specific promoters. While no significant differences in plant size, biomass or photosynthesis were found between PLC lines and wild-type (WT) plants, all PLC-OE lines, as well as those tissue-specific lines that promoted drought survival, exhibited a stronger decrease in convex hull perimeter (= increase in compactness) under water deprivation compared to WT. Increased compactness has not been associated with drought or decreased water loss before, though a hyponastic decrease in compactness in response to increased temperatures has been associated with water loss. We pose that increased compactness could lead to decreased water loss and potentially provides a new breeding trait to select for drought tolerance.
RESUMO
Several drought and salt tolerant phenotypes have been reported when overexpressing (OE) phospholipase C (PLC) genes across plant species. In contrast, a negative role for Arabidopsis PLC4 in salinity stress was recently proposed, showing that roots of PLC4-OE seedlings were more sensitive to NaCl while plc4 knock-out (KO) mutants were more tolerant. To investigate this apparent contradiction, and to analyse the phospholipid signalling responses associated with salinity stress, we performed root growth- and phospholipid analyses on plc4-KO and PLC4-OE seedlings subjected to salinity (NaCl) or osmotic (sorbitol) stress and compared these with wild type (WT). Only very minor differences between PLC4 mutants and WT were observed, which even disappeared after normalization of the data, while in soil, PLC4-OE plants were clearly more drought tolerant than WT plants, as was found earlier when overexpressing Arabidopsis PLC2, -3, -5, -7 or -9. We conclude that PLC4 plays no opposite role in salt-or osmotic stress and rather behaves like the other Arabidopsis PLCs.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Pressão Osmótica , Fosfolipídeos , Plantas Geneticamente Modificadas/genética , Plântula/metabolismo , Cloreto de Sódio/farmacologia , Estresse Fisiológico/genéticaRESUMO
Membrane fluidity, permeability, and surface charges are controlled by phospholipid metabolism and transport. Despite the importance of phosphatidic acid (PA) as a bioactive molecule, the mechanical properties of PA translocation and subcellular accumulation are unknown. Here, we used a mobilizable, highly responsive genetically encoded fluorescent indicator, green fluorescent protein (GFP)-N160RbohD, to monitor PA dynamics in living cells. The majority of GFP-N160RbohD accumulated at the plasma membrane and sensitively responded to changes in PA levels. Cellular, pharmacological, and genetic analyses illustrated that both salinity and abscisic acid rapidly enhanced GFP-N160RbohD fluorescence at the plasma membrane, which mainly depended on hydrolysis of phospholipase D. By contrast, heat stress induced nuclear translocation of PA indicated by GFP-N160RbohD through a process that required diacylglycerol kinase activity, as well as secretory and endocytic trafficking. Strikingly, we showed that gravity triggers asymmetric PA distribution at the root apex, a response that is suppressed by PLDζ2 knockout. The broad utility of the PA sensor will expand our mechanistic understanding of numerous lipid-associated physiological and cell biological processes and facilitate screening for protein candidates that affect the synthesis, transport, and metabolism of PA.
Assuntos
Ácidos Fosfatídicos , Fosfolipase D , Ácidos Fosfatídicos/análise , Ácidos Fosfatídicos/metabolismo , Membrana Celular/metabolismo , Transporte Biológico , Fosfolipase D/genética , Fosfolipase D/metabolismoRESUMO
We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.
Assuntos
Dióxido de Carbono , Mudança Climática , Estresse Fisiológico , Dióxido de Carbono/metabolismo , Transpiração Vegetal/fisiologia , Plantas/metabolismo , Água/metabolismoRESUMO
Pollen tubes require a tightly regulated pectin secretion machinery to sustain the cell wall plasticity required for polar tip growth. Involved in this regulation at the apical plasma membrane are proteins and signaling molecules, including phosphoinositides and phosphatidic acid (PA). However, the contribution of diacylglycerol kinases (DGKs) is not clear. We transiently expressed tobacco DGKs in pollen tubes to identify a plasma membrane (PM)-localized isoform, and then to study its effect on pollen tube growth, pectin secretion and lipid signaling. In order to potentially downregulate DGK5 function, we overexpressed an inactive variant. Only one of eight DGKs displayed a confined localization at the apical PM. We could demonstrate its enzymatic activity and that a kinase-dead variant was inactive. Overexpression of either variant led to differential perturbations including misregulation of pectin secretion. One mode of regulation could be that DGK5-formed PA regulates phosphatidylinositol 4-phosphate 5-kinases, as overexpression of the inactive DGK5 variant not only led to a reduction of PA but also of phosphatidylinositol 4,5-bisphosphate levels and suppressed related growth phenotypes. We conclude that DGK5 is an additional player of polar tip growth that regulates pectin secretion probably in a common pathway with PI4P 5-kinases.
Assuntos
Nicotiana , Tubo Polínico , Membrana Celular/metabolismo , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Fosfatidilinositóis/metabolismo , Nicotiana/metabolismoRESUMO
Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regiões de Interação com a Matriz , Antígenos de Histocompatibilidade Menor/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a low-abundance membrane lipid essential for plasma membrane function1,2. In plants, mutations in phosphatidylinositol 4-phosphate (PI4P) 5-kinases (PIP5K) suggest that PI(4,5)P2 production is involved in development, immunity and reproduction3-5. However, phospholipid synthesis is highly intricate6. It is thus likely that steady-state depletion of PI(4,5)P2 triggers confounding indirect effects. Furthermore, inducible tools available in plants allow PI(4,5)P2 to increase7-9 but not decrease, and no PIP5K inhibitors are available. Here, we introduce iDePP (inducible depletion of PI(4,5)P2 in plants), a system for the inducible and tunable depletion of PI(4,5)P2 in plants in less than three hours. Using this strategy, we confirm that PI(4,5)P2 is critical for various aspects of plant development, including root growth, root-hair elongation and organ initiation. We show that PI(4,5)P2 is required to recruit various endocytic proteins, including AP2-µ, to the plasma membrane, and thus to regulate clathrin-mediated endocytosis. Finally, we find that inducible PI(4,5)P2 perturbation impacts the dynamics of the actin cytoskeleton as well as microtubule anisotropy. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P2 in given cellular or developmental responses, and also to evaluate the importance of this lipid in protein localization.
Assuntos
Arabidopsis/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Inositol Polifosfato 5-Fosfatases/genética , Fosfatidilinositol 4,5-Difosfato/fisiologia , Fosfolipídeos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente ModificadasRESUMO
Membranes are essential for cells and organelles to function. As membranes are impermeable to most polar and charged molecules, they provide electrochemical energy to transport molecules across and create compartmentalized microenvironments for specific enzymatic and cellular processes. Membranes are also responsible for guided transport of cargoes between organelles and during endo- and exocytosis. In addition, membranes play key roles in cell signaling by hosting receptors and signal transducers and as substrates and products of lipid second messengers. Anionic lipids and their specific interaction with target proteins play an essential role in these processes, which are facilitated by specific lipid-binding domains. Protein crystallography, lipid-binding studies, subcellular localization analyses, and computer modeling have greatly advanced our knowledge over the years of how these domains achieve precision binding and what their function is in signaling and membrane trafficking, as well as in plant development and stress acclimation.
Assuntos
Transporte Biológico Ativo/fisiologia , Membrana Celular/metabolismo , Metabolismo dos Lipídeos , Fenômenos Fisiológicos Vegetais , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Plants alter their morphology and cellular homeostasis to promote resilience under a variety of heat regimes. Molecular processes that underlie these responses have been intensively studied and found to encompass diverse mechanisms operating across a broad range of cellular components, timescales and temperatures. This review explores recent progress throughout this landscape with a particular focus on thermosensing in the model plant Arabidopsis. Direct temperature sensors include the photosensors phytochrome B and phototropin, the clock component ELF3 and an RNA switch. In addition, there are heat-regulated processes mediated by ion channels, lipids and lipid-modifying enzymes, taking place at the plasma membrane and the chloroplast. In some cases, the mechanism of temperature perception is well understood but in others, this remains an open question. Potential novel thermosensing mechanisms are based on lipid and liquid-liquid phase separation. Finally, future research directions of high temperature perception and signalling pathways are discussed.
Assuntos
Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/fisiologia , Sensação Térmica/fisiologia , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Metabolismo dos Lipídeos , Fitocromo B/fisiologiaRESUMO
Plants adjust to unfavorable conditions by altering physiological activities, such as gene expression. Although previous studies have identified multiple stress-induced genes, the function of many genes during the stress responses remains unclear. Expression of ERD7 (EARLY RESPONSE TO DEHYDRATION 7) is induced in response to dehydration. Here, we show that ERD7 plays essential roles in both plant stress responses and development. In Arabidopsis, ERD7 protein accumulated under various stress conditions, including exposure to low temperature. A triple mutant of Arabidopsis lacking ERD7 and two closely related homologs had an embryonic lethal phenotype, whereas a mutant lacking the two homologs and one ERD7 allele had relatively round leaves, indicating that the ERD7 gene family has essential roles in development. Moreover, the importance of the ERD7 family in stress responses was evidenced by the susceptibility of the mutant lines to cold stress. ERD7 protein was found to bind to several, but not all, negatively charged phospholipids and was associated with membranes. Lipid components and cold-induced reduction in PIP2 in the mutant line were altered relative to wild type. Furthermore, membranes from the mutant line had reduced fluidity. Taken together, ERD7 and its homologs are important for plant stress responses and development and associated with the modification in membrane lipid composition.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Membrana Celular/metabolismo , Proteínas de Cloroplastos/fisiologia , Resposta ao Choque Frio , Lipídeos de Membrana/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/química , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Lipídeos de Membrana/análise , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipídeos/análise , Fosfolipídeos/metabolismoRESUMO
Phosphatidylinositol 3-phosphate (PI3P) is an essential membrane signature for both autophagy and endosomal sorting that is synthesized in plants by the class III phosphatidylinositol 3-kinase (PI3K) complex, consisting of the VPS34 kinase, together with ATG6, VPS15, and either VPS38 or ATG14 as the fourth subunit. Although Arabidopsis (Arabidopsis thaliana) plants missing the three core subunits are infertile, vps38 mutants are viable but have aberrant leaf, root, and seed development, Suc sensing, and endosomal trafficking, suggesting that VPS38 and ATG14 are nonredundant. Here, we evaluated the role of ATG14 through a collection of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and T-DNA insertion mutants disrupting the two Arabidopsis paralogs. atg14a atg14b double mutants were relatively normal phenotypically but displayed pronounced autophagy defects, including reduced accumulation of autophagic bodies and cargo delivery during nutrient stress. Unexpectedly, homozygous atg14a atg14b vps38 triple mutants were viable but showed severely compromised rosette development and reduced fecundity, pollen germination, and autophagy, consistent with a need for both ATG14 and VPS38 to fully actuate PI3P biology. However, the triple mutants still accumulated PI3P, but they were hypersensitive to the PI3K inhibitor wortmannin, indicating that the ATG14/VPS38 component is not essential for PI3P synthesis. Collectively, the ATG14/VPS38 mutant collection now permits the study of plants altered in specific aspects of PI3P biology.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas Relacionadas à Autofagia/genética , Mutação , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Proteínas de Transporte Vesicular/genética , Wortmanina/farmacologiaRESUMO
Polyamines, such as putrescine, spermidine and spermine (Spm), are low-molecular-weight polycationic molecules present in all living organisms. Despite their implication in plant cellular processes, little is known about their molecular mode of action. Here, we demonstrate that polyamines trigger a rapid increase in the regulatory membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2 ), and that this increase is required for polyamine effects on K+ efflux in Arabidopsis roots. Using in vivo 32 Pi -labelling of Arabidopsis seedlings, low physiological (µm) concentrations of Spm were found to promote a rapid PIP2 increase in roots that was time- and dose-dependent. Confocal imaging of a genetically encoded PIP2 biosensor revealed that this increase was triggered at the plasma membrane. Differential 32 Pi -labelling suggested that the increase in PIP2 was generated through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity rather than inhibition of a phospholipase C or PIP2 5-phosphatase activity. Systematic analysis of transfer DNA insertion mutants identified PIP5K7 and PIP5K9 as the main candidates involved in the Spm-induced PIP2 response. Using non-invasive microelectrode ion flux estimation, we discovered that the Spm-triggered K+ efflux response was strongly reduced in pip5k7 pip5k9 seedlings. Together, our results provide biochemical and genetic evidence for a physiological role of PIP2 in polyamine-mediated signalling controlling K+ flux in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Raízes de Plantas/metabolismo , Potássio/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Raízes de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Poliaminas/metabolismo , Poliaminas/farmacologia , Espermina/metabolismoRESUMO
Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis (Arabidopsis thaliana) PLD family consists of 12 members, and for some of these, a specific function in resistance toward a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLDγ1, but not its close homologs PLDγ2 and PLDγ3, is specifically involved in plant immunity. Genetic inactivation of PLDγ1 resulted in increased resistance toward the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea As pldγ1 mutant plants responded with elevated levels of reactive oxygen species to MAMP treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pldγ1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are not likely the cause for the enhanced immunity in the pldγ1 line. Instead, the plasma-membrane-attached PLDγ1 protein colocalized and associated with the BAK1-INTERACTING RECEPTOR-LIKE KINASES BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLDγ1 and BIR2 was further promoted upon MAMP treatment. Hence, we propose that PLDγ1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipases/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Botrytis/patogenicidade , Proteínas de Membrana/genética , Fosfolipase D/metabolismo , Fosfolipases/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Pseudomonas syringae/patogenicidade , Espécies Reativas de Oxigênio/metabolismoRESUMO
Strigolactones (SLs) represent a class of plant hormones that regulate developmental processes and play a role in the response of plants to various biotic and abiotic stresses. Both in planta hormonal roles and ex planta signalling effects of SLs are potentially interesting agricultural targets. In this review, we explore various aspects of SL function and highlight distinct areas of agriculture that may benefit from the use of synthetic SL analogues, and we identify possible bottlenecks. Our objective is to identify where the contributions of science and stakeholders are still needed to achieve harnessing the benefits of SLs for a sustainable agriculture of the near future.
Assuntos
Lactonas , Reguladores de Crescimento de Plantas , Compostos Heterocíclicos com 3 Anéis , Estresse FisiológicoRESUMO
Signal transduction in plants determines their successful adaptation to diverse stress factors. Our group employed suspension cells to study the phosphoinositide pathway, which is triggered by aluminium stress. We investigated about members of the PI-specific phospholipase C (PLC) family and evaluated their transcription profiles in Coffea arabica (Ca) suspension cells after 14days of culture when treated or not with 100µM AlCl3. The four CaPLC1-4 members showed changes in their transcript abundance upon AlCl3 treatment. The expression profiles of CaPLC1/2 exhibited a rapid and transitory increase in abundance. In contrast, CaPLC3 and CaPLC4 showed that transcript levels were up-regulated in short times (at 30s), while only CaPLC4 kept high levels and CaPLC3 was reduced to basal after 3h of treatment. CaPLC proteins were heterologously expressed, and CaPLC2 and CaPLC4 were tested for in vitro activity in the presence or absence of AlCl3 and compared to Arabidopsis PLC2 (AtPLC2). A crude extract was isolated from coffee cells. CaPLC2 showed a similar inhibition (30%) as in AtPLC2 and in the crude extract, while in CaPLC4, the activity was enhanced by AlCl3. Additionally, we visualized the yellow fluorescent protein PH domain of human PLCδ1 (YFP-PHPLCδ1) subcellular localization in cells that were treated or not with AlCl3. In non-treated cells, we observed a polar fluorescence signal towards the fused membrane. However, when cells were treated with AlCl3, these signals were disrupted. Finally, this is the first time that PLC activity has been shown to be stimulated in vitro by AlCl3.
Assuntos
Alumínio/toxicidade , Coffea/efeitos dos fármacos , Coffea/enzimologia , Proteínas de Plantas/metabolismo , Fosfolipases Tipo C/metabolismo , Arabidopsis , Coffea/genética , Perfilação da Expressão Gênica , Humanos , Proteínas de Plantas/genética , Transdução de Sinais , Estresse Fisiológico , Fosfolipases Tipo C/genéticaRESUMO
Polyamines, such as putrescine (Put), spermidine (Spd), and spermine (Spm), are low-molecular-weight polycationic molecules found in all living organisms. Despite the fact that they have been implicated in various important developmental and adaptative processes, their mode of action is still largely unclear. Here, we report that Put, Spd, and Spm trigger a rapid increase in the signaling lipid, phosphatidic acid (PA) in Arabidopsis seedlings but also mature leaves. Using time-course and dose-response experiments, Spm was found to be the most effective; promoting PA responses at physiological (low µM) concentrations. In seedlings, the increase of PA occurred mainly in the root and partly involved the plasma membrane polyamine-uptake transporter (PUT), RMV1. Using a differential 32Pi-labeling strategy combined with transphosphatidylation assays and T-DNA insertion mutants, we found that phospholipase D (PLD), and in particular PLDδ was the main contributor of the increase in PA. Measuring non-invasive ion fluxes (MIFE) across the root plasma membrane of wild type and pldδ-mutant seedlings, revealed that the formation of PA is linked to a gradual- and transient efflux of K+. Potential mechanisms of how PLDδ and the increase of PA are involved in polyamine function is discussed.
RESUMO
Phospholipase C (PLC) has been suggested to play important roles in plant stress and development. To increase our understanding of PLC signaling in plants, we have started to analyze knock-out (KO), knock-down (KD) and overexpression mutants of Arabidopsis thaliana, which contains nine PLCs. Earlier, we characterized PLC2, PLC3 and PLC5. Here, the role of PLC7 is functionally addressed. Promoter-GUS analyses revealed that PLC7 is specifically expressed in the phloem of roots, leaves and flowers, and is also present in trichomes and hydathodes. Two T-DNA insertion mutants were obtained, i.e., plc7-3 being a KO- and plc7-4 a KD line. In contrast to earlier characterized phloem-expressed PLC mutants, i.e., plc3 and plc5, no defects in primary- or lateral root development were found for plc7 mutants. Like plc3 mutants, they were less sensitive to ABA during stomatal closure. Double-knockout plc3 plc7 lines were lethal, but plc5 plc7 (plc5/7) double mutants were viable, and revealed several new phenotypes, not observed earlier in the single mutants. These include a defect in seed mucilage, enhanced leaf serration, and an increased tolerance to drought. Overexpression of PLC7 enhanced drought tolerance too, similar to what was earlier found for PLC3-and PLC5 overexpression. In vivo 32Pi-labeling of seedlings and treatment with sorbitol to mimic drought stress, revealed stronger PIP2 responses in both drought-tolerant plc5/7 and PLC7-OE mutants. Together, these results show novel functions for PLC in plant stress and development. Potential molecular mechanisms are discussed.
RESUMO
Phospholipase C (PLC) is a well-known signaling enzyme in metazoans that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate and diacylglycerol as second messengers involved in mutiple processes. Plants contain PLC too, but relatively little is known about its function there. The model system Arabidopsis thaliana contains nine PLC genes. Reversed genetics have implicated several roles for PLCs in plant development and stress signaling. Here, PLC5 is functionally addressed. Promoter-ß-glucuronidase (GUS) analyses revealed expression in roots, leaves and flowers, predominantly in vascular tissue, most probably phloem companion cells, but also in guard cells, trichomes and root apical meristem. Only one plc5-1 knock-down mutant was obtained, which developed normally but grew more slowly and exhibited reduced primary root growth and decreased lateral root numbers. These phenotypes could be complemented by expressing the wild-type gene behind its own promoter. Overexpression of PLC5 (PLC5-OE) using the UBQ10 promoter resulted in reduced primary and secondary root growth, stunted root hairs, decreased stomatal aperture and improved drought tolerance. PLC5-OE lines exhibited strongly reduced phosphatidylinositol 4-monophosphate (PIP) and PIP2 levels and increased amounts of phosphatidic acid, indicating enhanced PLC activity in vivo. Reduced PIP2 levels and stunted root hair growth of PLC5-OE seedlings could be recovered by inducible overexpression of a root hair-specific PIP 5-kinase, PIP5K3. Our results show that PLC5 is involved in primary and secondary root growth and that its overexpression improves drought tolerance. Independently, we provide new evidence that PIP2 is essential for the polar tip growth of root hairs.