RESUMO
Plants, as sessile organisms, have adapted a fine sensing system to monitor environmental changes, therefore allowing the regulation of their responses. As the interaction between plants and environmental changes begins at the surface, these changes are detected by components in the plasma membrane, where a molecule receptor generates a lipid signaling cascade via enzymes, such as phospholipases (PLs). Phospholipids are the key structural components of plasma membranes and signaling cascades. They exist in a wide range of species and in different proportions, with conversion processes that involve hydrophilic enzymes, such as phospholipase-C (PLC), phospholipase-D (PLD), and phospholipase-A (PLA). Hence, it is suggested that PLC and PLD are highly conserved, compared to their homologous genes, and have formed clusters during their adaptive history. Additionally, they generate responses to different functions in accordance with their protein structure, which should be reflected in specific signal transduction responses to environmental stress conditions, including innate immune responses. This review summarizes the phospholipid systems associated with signaling pathways and the innate immune response.
RESUMO
Plants are subject to different types of stress, which consequently affect their growth and development. They have developed mechanisms for recognizing and processing an extracellular signal. Second messengers are transient molecules that modulate the physiological responses in plant cells under stress conditions. In this sense, it has been shown in various plant models that membrane lipids are substrates for the generation of second lipid messengers such as phosphoinositide, phosphatidic acid, sphingolipids, and lysophospholipids. In recent years, research on lipid second messengers has been moving toward using genetic and molecular approaches to reveal the molecular setting in which these molecules act in response to osmotic stress. In this sense, these studies have established that second messengers can transiently recruit target proteins to the membrane and, therefore, affect protein conformation, activity, and gene expression. This review summarizes recent advances in responses related to the link between lipid second messengers and osmotic stress in plant cells.
Assuntos
Lipídeos/fisiologia , Pressão Osmótica/fisiologia , Plantas/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Cálcio/metabolismo , Glicolipídeos/fisiologia , Modelos Biológicos , Fosfolipídeos/fisiologia , Proteínas de Plantas/metabolismo , Estresse Salino/fisiologiaRESUMO
BACKGROUND: Mexico is considered the diversification center for chili species, but these crops are susceptible to infection by pathogens such as Colletotrichum spp., which causes anthracnose disease and postharvest decay in general. Studies have been carried out with isolated strains of Colletotrichum in Capsicum plants; however, under growing conditions, microorganisms generally interact with others, resulting in an increase or decrease of their ability to infect the roots of C. chinense seedlings and thus, cause disease. RESULTS: Morphological changes were evident 24 h after inoculation (hai) with the microbial consortium, which consisted primarily of C. ignotum. High levels of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA) were found around 6 hai. These metabolic changes could be correlated with high transcription levels of diacylglycerol-kinase (CchDGK1 and CchDG31) at 3, 6 and 12 hai and also to pathogen gene markers, such as CchPR1 and CchPR5. CONCLUSIONS: Our data constitute the first evidence for the phospholipids signalling events, specifically DGPP and PA participation in the phospholipase C/DGK (PI-PLC/DGK) pathway, in the response of Capsicum to the consortium, offering new insights on chilis' defense responses to damping-off diseases.
Assuntos
Capsicum/imunologia , Colletotrichum/fisiologia , Consórcios Microbianos/fisiologia , Fosfolipídeos/metabolismo , Doenças das Plantas/imunologia , Imunidade Vegetal , Transdução de Sinais , Capsicum/genética , Capsicum/microbiologia , Colletotrichum/isolamento & purificação , Diacilglicerol Quinase , Difosfatos/metabolismo , Glicerol/análogos & derivados , Glicerol/metabolismo , Interações Hospedeiro-Patógeno , Ácidos Fosfatídicos/metabolismo , Filogenia , Doenças das Plantas/microbiologia , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Raízes de Plantas/microbiologia , Plântula/genética , Plântula/imunologia , Plântula/microbiologia , Fosfolipases Tipo C/metabolismoRESUMO
Salicylic acid (SA) is an important signaling molecule involved in plant defense. While many proteins play essential roles in SA signaling, increasing evidence shows that responses to SA appear to involve and require lipid signals. The phospholipid-generated signal transduction involves a family of enzymes that catalyze the hydrolysis or phosphorylation of phospholipids in membranes to generate signaling molecules, which are important in the plant cellular response. In this review, we focus first, the role of SA as a mitigator in biotic/abiotic stress. Later, we describe the experimental evidence supporting the phospholipid-SA connection in plant cells, emphasizing the roles of the secondary lipid messengers (phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA)) and related enzymes (phospholipase D (PLD) and phospholipase C (PLC)). By placing these recent finding in context of phospholipids and SA in plant cells, we highlight the role of phospholipids as modulators in the early steps of SA triggered transduction in plant cells.
Assuntos
Ácidos Fosfatídicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Células Vegetais/metabolismo , Ácido Salicílico/farmacologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Fosfolipase D/metabolismo , Proteínas de Plantas/metabolismoRESUMO
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
Toxicity by aluminum is a growth-limiting factor in plants cultivated in acidic soils. This metal also promotes signal transduction pathways leading to the biosynthesis of defense compounds, including secondary metabolites. In this study, we observed that Coffea arabica L. cells that were kept in the dark did not produce detectable levels of caffeine. However, irradiation with light and supplementation of the culture medium with theobromine were the best conditions for cell maintenance to investigate the role of aluminum in caffeine biosynthesis. The addition of theobromine to the cells did not cause any changes to cell growth and was useful for the bioconversion of theobromine to caffeine. During a short-term AlCl3-treatment (500µM) of C. arabica cells kept under light irradiation, increases in the caffeine levels in samples that were recovered from both the cells and culture media were evident. This augmentation coincided with increases in the enzyme activity of caffeine synthase (CS) and the transcript level of the gene encoding this enzyme (CS). Together, these results suggest that actions by Al and theobromine on the same pathway lead to the induction of caffeine biosynthesis.
Assuntos
Alumínio/toxicidade , Cafeína/metabolismo , Coffea/efeitos dos fármacos , Células do Mesofilo/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Sementes/efeitos dos fármacos , Poluentes do Solo/toxicidade , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos da radiação , Linhagem Celular , Células Cultivadas , Coffea/citologia , Coffea/metabolismo , Coffea/efeitos da radiação , Meios de Cultivo Condicionados/química , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Células do Mesofilo/citologia , Células do Mesofilo/metabolismo , Células do Mesofilo/efeitos da radiação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas de Plantas/agonistas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos da radiação , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Sementes/citologia , Sementes/metabolismo , Sementes/efeitos da radiação , Teobromina/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiaçãoRESUMO
The protective effect of salicylic acid (SA) on aluminum (Al) toxicity was studied in suspension cells of Coffea arabica L. The results showed that SA does not produce any effect on cell growth and that the growth inhibition produced by aluminum is restored during simultaneous treatment of the cells with Al and SA. In addition, the cells exposed to both compounds, Al and SA, showed evident morphological signals of recovery from the toxic state produced in the presence of Al. The cells treated with SA showed a lower accumulation of Al, which was linked to restoration from Al toxicity because the concentration of Al(3+) outside the cells, measured as the Al(3+)-morin complex, was not modified by the presence of SA. Additionally, the inhibition of phospholipase C by Al treatment was restored during the exposure of the cells to SA and Al. The involvement of protein phosphorylation in the protective effect of SA on Al-toxicity was suggested because staurosporine, a protein kinase inhibitor, reverted the stimulatory effect of the combination of Al and SA on protein kinase activity. These results suggest that SA attenuates aluminum toxicity by affecting a signaling pathway linked to protein phosphorylation.
Assuntos
Alumínio/farmacologia , Coffea/metabolismo , Proteínas de Plantas/metabolismo , Ácido Salicílico/farmacologia , Alumínio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Coffea/citologia , Coffea/ultraestrutura , Relação Dose-Resposta a Droga , Antagonismo de Drogas , Inibidores Enzimáticos/farmacologia , Microscopia Eletrônica de Varredura , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Fatores de Tempo , Fosfolipases Tipo C/metabolismoRESUMO
Measurement of phospholipase C (PLC) activity in vitro is a valuable biochemistry technique easily applicable in samples from different organisms. It quantifies the enzymatic activity of a key protein involved in critical developmental functions in organisms such as plants, animals, and bacteria. A protocol is described which assays the formation of two main products of the PLC hydrolysis reaction on radioactively labeled phospholipid substrates.
Assuntos
Ensaios Enzimáticos/métodos , Fosfolipases Tipo C/metabolismo , Animais , Fosfatidilinositol 4,5-Difosfato/metabolismo , Especificidade por Substrato , TrítioRESUMO
There is limited information about the involvement of lipids and esterified fatty acids in signaling pathways during plant development. The purpose of this study was to evaluate the lipid composition and molecular species of potato (Solanum tuberosum L., cv. Spunta) stolons and to identify phosphorylated lipids in the first two developmental stages of tuber formation. Lipid profiling was determined using ESI-MS/MS, a useful method for the determination of the biosynthesis and catabolism of lipids based on their fatty acid composition. The most prevalent compound identified in this study was phosphatidic acid (PA); digalactosyldiacylglycerol (DGDG) was the second most abundant compound. A 34:2 species was identified in PA, phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). The identification of lipid phosphorylation by kinases was revealed by the presence of the phosphorylated lipids. PA was metabolized to diacylglycerol pyrophosphate (DGPP) by phosphatidic acid kinase (PAK). This work establishes a correlation between lipid fatty acid composition and lipid metabolism enzymes at the beginning of tuber formation and is the first report of PAK activity in the early events of potato tuber formation.
Assuntos
Metabolismo dos Lipídeos , Fosfotransferases/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Fosforilação , Tubérculos/química , Tubérculos/enzimologia , Tubérculos/crescimento & desenvolvimento , Tubérculos/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/metabolismoRESUMO
The accumulation of reactive oxygen species (ROS) and concomitant oxidative stress have been considered deleterious consequences of aluminum toxicity. However, several lines of evidence suggest that ROS can function as important signaling molecules in the plant defense system for protection from abiotic stress and the acquisition of tolerance. The role of ROS-scavenging enzymes was assayed in two different coffee cell suspension lines. We treated L2 (Al-sensitive) and LAMt (Al-tolerant) Coffea arabica suspension cells with 100 µM AlCl(3) and observed significant differences in catalase activity between the two cell lines. However, we did not observe any differences in superoxide dismutase or glutathione reductase activity in either cell line following Al treatment. ROS production was diminished in the LAMt cell line. Taken together, these results indicate that aluminum treatment may impair the oxidative stress response in L2 cells but not in LAMt cells. We suggest a possible role for Al-induced oxidative bursts in the signaling pathways that lead to Al resistance and protection from Al toxicity.
Assuntos
Catalase/metabolismo , Coffea/citologia , Glutationa Redutase/metabolismo , Proteínas de Plantas/metabolismo , Superóxido Dismutase/metabolismo , Técnicas de Cultura de Células , Coffea/efeitos dos fármacos , Coffea/enzimologia , Tolerância a Medicamentos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
The phospholipidic signal transduction system involves generation of second messengers by hydrolysis or changes in phosphorylation state. Several studies have shown that the signaling pathway forms part of plant response to phytoregulators such as salicylic acid (SA) and methyl jasmonate (MJ), which have been widely used to stimulate secondary metabolite production in cell cultures. An evaluation was made of the effect of SA and MJ on phospholipidic signaling and capsaicinoid production in Capsicum chinense Jacq. suspension cells. Treatment with SA inhibited phospholipase C (PLC) (EC: 3.1.4.3) and phospholipase D (PLD) (EC: 3.1.4.4) activities in vitro, but increased lipid kinase activities in vitro at different SA concentrations. Treatment with MJ produced increases in PLC and PLD activities, while lipid kinase activities were variable and dose-dependent. The production of vanillin, a precursor of capsaicinoids, increased at specific SA or MJ doses. Preincubation with neomycin, a phospholipase inhibitor, before SA or MJ treatment inhibits increase in vanillin production which suggests that phospholipidic second messengers may participate in the observed increase in vanillin production.
Assuntos
Acetatos/farmacologia , Benzaldeídos/metabolismo , Capsicum/metabolismo , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Fosfolipídeos/metabolismo , Ácido Salicílico/farmacologia , Capsicum/efeitos dos fármacos , Capsicum/enzimologia , Células Cultivadas , Fosfolipase D/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
Biotechnological advances in coffee research (in vitro manipulation, multiplication, generation and development of transgenic coffee plants with specific traits like high yield and good quality) have contributed to description of the metabolic pathways involved in the response mechanisms to environmental factors like abiotic stress. Coffea arabica L. plants grow in acidic soils, and therefore aluminium (Al) toxicity is a major negative impact on crop productivity. To understand Al toxicity mechanisms in cells via the Al absorption kinetic, we isolated protoplasts from two C. arabica L. suspension cell lines: Al-sensitive (L2) and Al-tolerant (LAMt). Protoplasts of LAMt line exhibited lower Al absorption levels than protoplasts of the L2 line. Use of two fluorescent tracers (morin and calcofluor white) indicated that Al interacts with internal cell structures, such as the plasma membrane and nucleus, with differences in both cell lines. Al-tolerance in the LAMt is probably associated with the cell wall as well as intracellular structures. These data will help to better understand Al toxicity in C. arabica, and Al toxicity mechanisms in plant cells.
Assuntos
Alumínio/metabolismo , Coffea/metabolismo , Protoplastos/metabolismo , Alumínio/análise , Alumínio/toxicidade , Células Cultivadas , Coffea/citologia , Coffea/efeitos dos fármacos , Protoplastos/efeitos dos fármacosRESUMO
In acid soils, aluminium (Al) toxicity and phosphate (Pi) deficiency are the most significant constraints on plant growth. Al inhibits cell growth and disrupts signal transduction processes, thus interfering with metabolism of phospholipase C (PLC), an enzyme involved in second messenger production in the cell. Using a Coffea arabica suspension cell model, we demonstrate that cell growth inhibition by Al toxicity is mitigated at a high Pi concentration. Aluminium-induced cell growth inhibition may be due to culture medium Pi deficiency, since Pi forms complexes with Al, reducing Pi availability to cells. Phosphate does not mitigate inhibition of PLC activity by Al toxicity. Other enzymes of the phosphoinositide signal transduction pathway were also evaluated. Aluminium disrupts production of second messengers such as inositol 1,4,5-trisphosphate (IP(3)) and phosphatidic acid (PA) by blocking PLC activity; however, phospholipase D (PLD) and diacylglycerol kinase (DGK) activities are stimulated by Al, a response probably aimed at counteracting Al effects on PA formation. Phosphate deprivation also induces PLC and DGK activity. These results suggest that Al-induced cell growth inhibition is not linked to PLC activity inhibition.
Assuntos
Alumínio/farmacologia , Coffea/efeitos dos fármacos , Fosfatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Alumínio/análise , Células Cultivadas , Coffea/crescimento & desenvolvimento , Coffea/metabolismo , Diacilglicerol Quinase/efeitos dos fármacos , Diacilglicerol Quinase/metabolismo , Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Inositol 1,4,5-Trifosfato/metabolismo , Ácidos Fosfatídicos/antagonistas & inibidores , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/efeitos dos fármacos , Fosfolipase D/metabolismo , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
The primary Al-tolerance mechanism in plants involves exudation and/or accumulation of specific organic acid species, which form non-phytotoxic complexes with Al(3+) under physiological conditions. An evaluation was done of the role of organic acids in the tolerance mechanism of a cell suspension line of coffee Coffea arabica that exhibits Al-tolerance (LAMt) but for which the metabolic tolerance mechanism remains unknown. Significant differences existed in malate dehydrogenase and citrate synthase activities (key enzymes in organic acids metabolism) between protein extracts (day 7 of culture cycle) of the L2 (Al-sensitive) and LAMt (Al-tolerant) cells when cell suspensions were treated with 100 microM AlCl(3). HPLC analysis showed that the suspension cells of both lines exudate malate when incubated in a minimal solution but that exudation was not enhanced by treatment with AlCl(3) (100 microM). This is the first study demonstrating that plant Al-tolerance may be associated with down-regulation of malate dehydrogenase and citrate synthase activities.
Assuntos
Alumínio/farmacologia , Ácidos Carboxílicos/metabolismo , Coffea/metabolismo , Tolerância a Medicamentos , Citrato (si)-Sintase/efeitos dos fármacos , Coffea/citologia , Coffea/efeitos dos fármacos , Coffea/enzimologia , Regulação para Baixo/efeitos dos fármacos , Malato Desidrogenase/efeitos dos fármacos , MalatosRESUMO
Coffee (Coffea arabica L.) is of economic importance worldwide. Its growth in organic-rich acidic soils is influenced by aluminium such that coffee yield may be impaired. Herein we have used the Al-sensitive C. arabica suspension cell line L2 to analyse the effect of two different Al species on the phosphoinositide signal transduction pathway. Our results have shown that the association of Al with coffee cells was affected by the pH and the form of Al in media. More Al was associated with cells at pH 4.3 than 5.8, whereas when Al was present as hydroxyaluminosilicates (HAS) the association was halved at pH 4.3 and unchanged at pH 5.8. Two signal transduction elements were also evaluated; phospholipase C (PLC) activity and phosphatidic acid (PA) formation. PLC was inhibited ( approximately 50%) when cells were incubated for 2 h in the presence of either AlCl(3) or Al in the form of HAS. PA formation was tested as a short-term response to Al. By way of contrast to what was found for PLC, incubation of cells for 15 min in the presence of AlCl(3) decreased the formation of PA whereas the same concentration of Al as HAS produced no effect upon its formation. These results suggest that Al is capable to exert its effects upon signal transduction as Al((aq))(3+) acting upon a mechanism linked to the phosphoinositide signal transduction pathway.
Assuntos
Alumínio/antagonistas & inibidores , Alumínio/toxicidade , Coffea/efeitos dos fármacos , Coffea/metabolismo , Fosfolipídeos/metabolismo , Ácido Silícico/farmacologia , Linhagem Celular , Concentração de Íons de Hidrogênio , Inositol 1,4,5-Trifosfato/metabolismo , Ácidos Fosfatídicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
Recent results, fundamentally obtained from animal tissues, suggest that polyamines (Pas), essential compounds for the growth and development of all life organisms, may interact with a signal transduction cascade. Because Pas are highly positive charged compounds, their binding with phospholipids involved in signal transduction is likely to be the case. In this work, the in vivo effect of Pas on some important components of phospholipid signal transduction pathway was studied, by the first time, in plant tissue. Endogenous Pas content varied during the culture cycle of Coffea arabica cells: putrescine (Put) levels increased at the end of the stationary phase, both spermidine (Spd) and spermine (Spm) accumulated at the beginning of the linear growth phase. Cells that were incubated with Put presented a significant increase in phospholipase D (PLD) (EC: 3.1.4.4) activity, phospholipase C (PLC) (EC: 3.1.4.3) activity decreased, and the effect on lipid kinases was less marked. However, the incubation of the cells with Spd and Spm significantly stimulated the lipid kinases activities, fundamentally increased the formation of phosphatidyl inositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2), while the effect on PLC and PLD activities was minor when compared with the cells treated with Put. The results presented here suggest that Pas may modulate the cellular signal of C. arabica cells by differentially affecting components of the phospholipid cascade.
Assuntos
Poliaminas Biogênicas/fisiologia , Coffea/metabolismo , Fosfolipídeos/metabolismo , Transdução de Sinais/fisiologia , Coffea/citologia , Coffea/enzimologia , Fosfolipase D/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
An aluminium (Al)-tolerant cell line (LAMt) of coffee (Coffea arabica L.) was obtained from a cell suspension culture and biochemically and molecularly characterized in an MS medium at half ionic strength and low pH. LAMt grew 30% more than the control line (susceptible to Al) in the presence of different concentrations of Al, showed a lower free Al concentration in the medium and had higher phospholipase C specific activity (80%). Membrane integrity of the LAMt was 50% greater than the control line when both were incubated in the presence of different Al concentrations (measured by Evans Blue uptake). Finally, the use of microsatellite primers revealed no difference in the DNA pattern of both cell lines.