Effect of phosphate on aluminium-inhibited growth and signal transduction pathways in Coffea arabica suspension cells

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 (IP₃) 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.     

Aluminium differentially modifies lipid metabolism from the phosphoinositide pathway in Coffea arabica cells

The effect of aluminium (Al) on phosphoinositide-specific phospholipase C (PLC) and lipid kinase activities was examined in a cellular suspension of coffee. Two main effects were seen when cells were treated with AlCl3. In periods as short as 1 minute, Al-exposed cells increased the activity of PLC and IP3 formation up to two fold. Over longer periods PLC activity was inhibited by more than 50%. The activity of phosphatidylinositol 4-kinase (Pl 4-K), phosphatidylinositol phosphate 5-kinase (PIP 5-K) and diacylglycerol kinase increased when cells were incubated in the presence of different concentrations of AlCl3. The present study reports for the first time that Al may have different effects on the Pl-signaling pathway depending on the time of exposure. Our results strongly support the hypothesis that Al disrupts the metabolism of membrane phospholipids regulating not only PLC but also other enzymes that have key roles in signal-transduction pathways.     

Aluminum Inhibition of the Inositol 1,4,5-Trisphosphate Signal Transduction Pathway in Wheat Roots: A Role in Aluminum Toxicity?

In crop plants, aluminum (Al) rhizotoxicity is a major problem worldwide; however, the cause of Al toxicity remains elusive. The effects of Al on the inositol 1,4,5-trisphosphate (Ins[1,4,5]P3)-mediated signal transduction pathway were investigated in wheat roots. Exogenously applied Al (50 [mu]M) rapidly inhibited root growth (<2 hr) but did not affect general root metabolism. An Ins(1,4,5)P3 transient was generated in root tips, either before or after exposure to Al for 1 hr, by treating the roots with H2O2 (10 mM). Background (unstimulated) levels of Ins(1,4,5)P3 were similar in both Al-treated and Al-untreated root apices. However, H2O2-stimulated levels of Ins(1,4,5)P3 in root apices showed a significant (>50%) reduction after Al exposure in comparison with untreated controls, indicating that Al may be interfering with the phosphoinositide signaling pathway. When phospholipase C (PLC) was assayed directly in the presence of Al or other metal cations in microsomal membranes, AlCl3 and Al-citrate specifically inhibited PLC action in a dose-dependent manner and at physiologically relevant Al levels. Al exposure had no effect on inositol trisphosphate dephosphorylation or on a range of enzymes isolated from wheat roots, suggesting that Al exposure may specifically target PLC. Possible mechanisms of PLC inhibition by Al and the role of Ins(1,4,5)P3 in Al toxicity and growth are discussed. This study provides compelling evidence that the phytotoxic metal cation Al has an intracellular target site that may be integrally involved in root growth.     

Polyamines modify the components of phospholipids-based signal transduction pathway in Coffea arabica L. cells.

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.     

Modification of the culture medium to produce aluminum toxicity in cell suspensions of coffee (Coffea arabica L.)

Coffee (Coffea arabica) plants are usually grown in soils containing high levels of organic materials. Under these conditions, aluminum (Al) is toxic because of the acidic nature of the soils. Al is the most abundant metal found in the earth's crust and occurs in a number of different forms in soil. In acid soils, Al toxicity is a global problem that limits crop productivity. A major problem in obtaining cellular lines displaying Al tolerance in culture is the composition of the medium. In the experiments presented here, we modified the composition of the culture medium for a C. arabica cell line to produce Al toxicity. Murashige-Skoog media was used, complete (MS) and half ionic strength (MSHIS), at either pH 5.8 or 4.3. We found that MSHIS and pH 4.3 provided the optimal conditions to obtain Al toxicity as measured by the ability to grow in a range of Al concentrations (25-1,000 µM). The lethal dose (LD₅₀) under these conditions was 25 µM. The concentrations of free Al in the culture medium were corroborated by the fluorescent compound Morin. Al was found to enter the cell after 30 min, and the signal was then retained for up to 2 h.     

Direct and indirect identification of the formation of hydroxyaluminosilicates in acidic solutions.

Morin-aluminium fluorescence and membrane filtration were successfully applied to the indirect identification of the formation of hydroxyaluminosilicates (HAS) in acidic solutions of varying pH and of known concentrations of aluminium (Al) and silicic acid (Si(OH)(4)). It was proven to be especially useful in providing evidence of the strong competition between Si(OH)(4) and Al(OH)(3) to condense with hydroxyaluminium templates to form HAS in preference to Al(OH)(3(s)). The aggregation and stability of HAS and Al(OH)(3(s)) were dependent upon both the pH and the [Al] of the solution. The applicability of these indirect techniques was confirmed using the direct observation of HAS in solution by atomic force microscopy (AFM). AFM was also a powerful tool in providing valuable information on the morphology of colloidal HAS of various structures and stoichiometries. The results have provided further confirmation of both the mechanism of HAS formation and the form and stability of HAS in solution. This information is essential to our understanding of the biological availability and hence toxicity of Al in biota, including man.     

Aluminum inhibits phosphatidic acid formation by blocking the phospholipase C pathway

Aluminum (Al³⁺) has been recognized as a main toxic factor in crop production in acid lands. Phosphatidic acid (PA) is emerging as an important lipid signaling molecule and has been implicated in various stress-signaling pathways in plants. In this paper, we focus on how PA generation is affected by Al³⁺ using Coffea arabica suspension cells. We pre-labeled cells with [³²P]orthophosphate (³²Pi) and assayed for ³²P-PA formation in response to Al³⁺. Treating cells for 15 min with either AlCl₃ or Al(NO₃)₃ inhibited the formation of PA. In order to test how Al³⁺ affected PA signaling, we used the peptide mastoparan-7 (mas-7), which is known as a very potent stimulator of PA formation. The Al³⁺ inhibited mas-7 induction of PA response, both before and after Al³⁺ incubation. The PA involved in signaling is generated by two distinct phospholipid signaling pathways, via phospholipase D (PLD; EC: 3.1.4.4) or via Phospholipase C (PLC; EC: 3.1.4.3), and diacylglycerol kinase (DGK; EC 2.7.1.107). By labeling with ³²Pi for short periods of time, we found that PA formation was inhibited almost 30% when the cells were incubated with AlCl₃ suggesting the involvement of the PLC/DGK pathway. Incubation of cells with PLC inhibitor, U73122, affected PA formation, like AlCl₃ did. PLD in vivo activation by mas-7 was reduced by Al³⁺. These results suggest that PA formation was prevented through the inhibition of the PLC activity, and it provides the first evidence for the role of Al toxicity on PA production.     

G Proteins Coupled to Phospholipase C: Molecular Targets of Long-Term Ethanol Exposure

Long-term ethanol exposure is known to inhibit bradykinin-stimulated phosphoinositide hydrolysis in cultures of neuroblastoma x glioma 108-15 cells. In the present study, [3H]bradykinin binding, GTP-binding protein function, and phospholipase C activity were assayed in cells grown for 4 days in 100 mM ethanol with the aim of elucidating the molecular target of ethanol on signal transduction coupled to inositol trisphosphate and diacylglycerol formation. Ethanol exposure reduced guanosine 5'-O-(3-thiotriphosphate) [GTP(S)]- and, to a lesser extent, NaF/AlCl3-stimulated phosphoinositide hydrolysis, whereas it had no effect on the enzymatic activity of a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. [3H]Bradykinin binding in the absence of GTP(S) was not influenced by ethanol exposure. However, the reduction in [3H]bradykinin binding seen in control cells after addition of GTP analogue was inhibited in cells grown in ethanol-containing medium. The results indicate that long-term ethanol exposure exerts its effects on receptor-stimulated phosphoinositide hydrolysis primarily at the level of the GTP-binding protein.     

Aluminum stress and its role in the phospholipid signaling pathway in plants and possible biotechnological applications

An early response of plants to environmental signals or abiotic stress suggests that the phospholipid signaling pathway plays a pivotal role in these mechanisms. The phospholipid signaling cascade is one of the main systems of cellular transduction and is related to other signal transduction mechanisms. These other mechanisms include the generation of second messengers and their interactions with various proteins, such as ion channels. This phospholipid signaling cascade is activated by changes in the environment, such as phosphate starvation, water, metals, saline stres, and plant-pathogen interactions. One important factor that impacts agricultural crops is metal-induced stress. Because aluminum has been considered to be a major toxic factor for agriculture conducted in acidic soils, many researchers have focused on understanding the mechanisms of aluminum toxicity in plants. We have contributed the last fifteen years in this field by studying the effects of aluminum on phospholipid signaling in coffee, one of the Mexico's primary crops. We have focused our research on aluminum toxicity mechanisms in Coffea arabica suspension cells as a model for developing future contributions to the biotechnological transformation of coffee crops such that they can be made resistant to aluminum toxicity. We conclude that aluminum is able to not only generate a signal cascade in plants but also modulate other signal cascades generated by other types of stress in plants. The aim of this review is to discuss possible involvement of the phospholipid signaling pathway in the aluminum toxicity response of plant cells.     

Signal transduction events in aluminum-induced cell death in tomato suspension cells

In this study, some of the signal transduction events involved in AlCl(3)-induced cell death in tomato (Lycopersicon esculentum Mill.) suspension cells were elucidated. Cells treated with 100 microM AlCl(3) showed typical features of programmed cell death (PCD) such as nuclear and cytoplasmic condensation. Cell death was effectively inhibited by protease and human caspase inhibitors indicating a cell death execution mechanism with similarities to animal apoptosis. Cell death was suppressed by application of antoxidants and by inhibitors of phospholipase C (PLC), phospholipase D (PLD) and ethylene signalling pathways. The results suggest that low concentrations of heavy metal ions stimulate both PLC and PLD signalling pathways leading to the production of reactive oxygen species (ROS) and subsequent cell death executed by caspase-like proteases.     

酸性环境下铝对斑马鱼运动行为和学习记忆能力的影响暨与阿尔海默茨症的相互关系

运用行为学方法通过金属离子诱发神经毒性,建立阿尔海默茨症动物模型。通过运动行为观察、应激回避条件模型检测在pH值为7.8、6.8、5.8条件下暴露铝离子24h和96h后铝离子对成年斑马鱼运动行为和学习记忆能力的作用,探讨金属元素在酸性环境下诱发神经毒性导致阿尔海默茨症与运动行为、学习记忆的关系。结果表明,pH5.8铝离子组暴露96h的运动行为活性和学习记忆能力与pH7.8铝离子组和pH6.8铝离子组相比有较显著变化。同时,pH5.8铝离子组暴露96h运动行为活性和学习记忆能力与pH5.8铝离子组暴露24h相比出现明显降低。这些都表明,铝在酸性环境下,与pH相互作用影响斑马鱼的运动行为与学习记忆能力,可能造成斑马鱼大脑关于记忆功能区域出现损伤,诱发神经毒性产生类似阿尔海默茨症发病症状。     

Cell population-based model of dermal wound invasion with heterogeneous intracellular signaling properties

A deterministic model of dermal wound invasion, which accounts for the platelet-derived growth factor (PDGF) gradient sensing mechanism in fibroblasts mediated by cell surface receptors and the phosphoinositide 3-kinase (PI3K) signal transduction pathway, was previously described (Biophys J 2006; 90:2297–308). Here, we extend that work and implement a hybrid modeling strategy that treats fibroblasts as discrete entities endowed with heterogeneous properties, namely receptor, PI3K and 3′ phosphoinositide phosphatase expression levels. Analysis of the model suggests that the wound environment fosters the advancement of cells within the population that are better fit to migrate and/or proliferate in response to PDGF stimulation. Thus, cell-to-cell variability results in a significantly higher rate of wound invasion as compared with the deterministic model, in a manner that depends on the way in which individual cell properties are sampled or inherited upon cell division.Key words: wound healing, chemotaxis, gradient sensing, mathematical model, stochastic, signal transduction, phosphoinositide 3-kinase, PDGF     

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.     

Changes in some characteristics between the wild and Al-tolerant coffee (Coffea arabica L.) cell line

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.     

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide

Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant-pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosan-induced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed.     

Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor

In this paper we demonstrate that cytoskeletons isolated from A431 cells have associated with them high activities of several kinases involved in inositol lipid metabolism, such as phosphatidylinositol kinase, phosphatidylinositol phosphate kinase, and diacylglycerol kinase. In addition also phospholipase C activity was detected on isolated cytoskeletons. Controlled extraction of the cytoskeletons followed by in vitro polymerization of actin demonstrated an association of the kinases to the actin filament system consisting of actin and a number of actin-binding proteins. The cytoskeleton-associated lipid kinase activities were significantly increased upon treatment of intact cells with EGF. These data suggest that the association of the phosphoinositide kinases, diacylglycerol kinase, phospholipase C, and also the EGF receptor to the cytoskeleton may play a role in the efficient signal transduction induced by EGF, by providing a matrix for the various components involved in signal transduction.     

Different expression and subcellular localization of Phosphoinositide-specific Phospholipase C enzymes in differently polarized macrophages

Macrophages’ phenotypic and functional diversity depends on differentiating programs related to local environmental factors. Recent interest was deserved to the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. The expression panel of PLCs and the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), as well as in M1 and M2 macrophages to list the expressed isoforms and their subcellular localization. Furthermore, we investigated whether inflammatory stimulation modified the basal panel of PLCs’ expression and subcellular localization. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes’ mRNA expression and intracellular localization of PLC enzymes. The panel of PLC genes’ expression and PLC proteins’ presence slightly changes after inflammatory stimulation. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization.     

Animal cell shape changes and gene expression.

Cell shape and cell contacts are determined by transmembrane receptor-mediated associations of the cytoskeleton with specific extracellular matrix proteins and with ligands on the surface of adjacent cells. The cytoplasmic domains of these microfilament-membrane associations at the adherens junction sites, also localize a variety of regulatory molecules involved in signal transduction and gene regulation. The stimulation of cells with soluble polypeptide factors leads to rapid changes in cell shape and microfilament component organization. In addition, this stimulation also activates the phosphoinositide signaling pathway. Recently, a linkage between actin-binding proteins and the phosphoinositide signaling pathway, was discovered. It is suggested that by the association with the second messenger system, and/or by controlling the localization of regulatory molecules, the cytoskeleton may regulate gene expression.     

Cell population-based model of dermal wound invasion with heterogeneous intracellular signaling properties

A deterministic model of dermal wound invasion, which accounts for the platelet-derived growth factor (PDGF) gradient sensing mechanism in fibroblasts mediated by cell surface receptors and the phosphoinositide 3-kinase (PI3K) signal transduction pathway, was previously described (Biophys J 2006; 90:2297-2308). Here, we extend that work and implement a hybrid modeling strategy that treats fibroblasts as discrete entities endowed with heterogeneous properties, namely receptor, PI3K and 3’ phosphoinositide phosphatase expression levels. Analysis of the model suggests that the wound environment fosters the advancement of cells within the population that are better fit to migrate and/or proliferate in response to PDGF stimulation. Thus, cell-to-cell variability results in a significantly higher rate of wound invasion as compared with the deterministic model, in a manner that depends on the way in which individual cell properties are sampled or inherited upon cell division.     

Binding of GM-CSF to adherent neutrophils activates phospholipase D.

When the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor was incubated with neutrophils adherent to plastic tissue culture plates or plates coated with extracellular matrix proteins, a rapid (3 min) but transient formation of phosphatidic acid was observed. This stimulation was dependent on the dose of GM-CSF, with an EC50 of 140 pM, and was further enhanced (up to 350%) with the PA phosphatase inhibitor propranolol in a dose-dependent manner. Conversely, GM-CSF was unable to trigger any PA formation in neutrophils maintained in suspension, even in the presence of soluble fibronectin. However, GM-CSF did prime the cells for enhanced PA formation in the presence of a secondary stimulus (fMet-Leu-Phe or PAF). GM-CSF also caused a time-dependent stimulation of diacylglycerol formation in adherent, but not suspended, cells and elicited a time-dependent stimulation of phosphatidylethanol formation, with a concomitant decrease in the formation of PA only at early (< 7 min) times. These observations were consistent with a rapid activation of the enzyme phospholipase D in adherent cells stimulated with GM-CSF. Additional data indicated that the source of DAG was PLD coexisting with PLC, especially at later times ( > 7 min) of stimulation with GM-CSF. Finally, the formation of PA and PEt, and to a minor extent, DAG, were inhibited by the protein tyrosine kinase inhibitor erbstatin in conditions in which tyrosine phosphorylation occurred. Taken together the data indicate that GM-CSF rapidly activates PLD in adherent cells, which is responsible for the generation of PA. Thus, PLD activation is an early event in neutrophil signal transduction following exposure of adherent cells to GM-CSF.